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Abstract:

A synthesis procedure for benzazolo[3,2-a]quinolinium chloride salts and
the inclusion of chloro-substituent, amino-substituent, and
nitro-substituent resulting in several compounds wherein said procedures
provides an increment in the compounds biological activity. The compounds
are further used for intra cellular binding, cytotoxicity on malignant
cells through apoptosis activation mediated by mitochondrial damage,
cellular organelles binding and damage, DNA fragmentation, marker of
bacterial growth, antibacterial activity, cell cycle disruption, and a
marker due to the auto-fluorescent properties.

Claims:

1. A synthesis procedure for a amino-substituted
benzazolo[3,2-a]quinolinium chloride salt compound consisting of
##STR00001## wherein R1 is 3NH2, R2 is selected from a
group consisting of H, 10-Me, 9-OMe, 8,9-C4H4, 10-OMe,
8,9-di-OMe, 10-OMe, 9-OMe, and X is selected from a group consisting of
NCH2C6H5, S, N-n-Butyl, NMe, NiPr, NCH═CHCH3, and
c-C5H9, comprising the steps of: a. synthesis of a
nitro-substituted 2-(2-chlorostyryl)benzazole; b. reduction of the nitro
group to form an amino-substituted 2-(2-chlorostyryl)benzazole; and c.
photo-induced cyclization procedure of said amino-substituted
2-(2-chlorostyryl)benzazole to generate a biological florescent
amino-substituted benzazolo[3,2-a]quinolinium chloride salt.

2. A synthesis procedure for a amino-substituted
benzazolo[3,2-a]quinolinium chloride salts compound as in claim 1 wherein
the reduction of nitro group comprises a hydrazine using nickel boride as
catalyst.

3. A synthesis procedure for an amino-substituted
benzazolo[3,2-a]quinolinium chloride salt compound as in claim 1 wherein
the photo-induced cyclization procedure comprises: a. placing a preferred
amount of a 2-(2-chlorostyryl)benzazole inside a vessel; and b.
dissolving said preferred amount in a 2:2:1 heptane:bromobenzene:dioxane
mixture.

4. A synthesis procedure for an amino-substituted
benzazolo[3,2-a]quinolinium chloride salt compound as in claim 3 wherein
the photo-induced cyclization procedure comprises: a. placing 0.2-1.0 g
of said 2-(2-chlorostyryl)benzazole inside said vessel; and b. dissolving
said 2-(2-chlorostyryl)benzazole in 150-250 mL of 2:2:1
heptane:bromobenzene:dioxane mixture.

5. A synthesis procedure for a chloro-substituted
benzazolo[3,2-a]quinolinium chloride salt compound consisting of
##STR00002## wherein R1 is selected from a group consisting of
4-Cl, 2,3-diCl, H, R2 is H, R3 is selected from a group
consisting of H, 10-CH3, and 9-OCH3, and X is S, comprising the
steps of: a. synthesis of a chloro-substituted
2-(2-chlorostyryl)benzazole; b. photo-induced cyclization procedure of
said chloro-substituted 2-(2-chlorostyryl)benzazole to generate a
biological florescent chloro-substituted benzazolo[3,2-a]quinolinium
chloride salt.

6. A method of using the biological activity of
benzazolo[3,2-a]quinolinium chloride salts comprising: a
benzazolo[3,2-a]quinolinium chloride salt and at least one cell wherein
said cell is exposed to a selected concentration of
benzazolo[3,2-a]quinolinium chloride, wherein said selected amount
modifies the biological activity of said cell.

7. A method of using the biological activity of
benzazolo[3,2-a]quinolinium chloride salts as in claim 6 wherein the
selected concentration of the benzazolo[3,2-a]quinolinium chloride salt
is within the range bounded by the IC25 and the IC90 of said
cell.

8. A method of using the biological activity of
benzazolo[3,2-a]quinolinium chloride salts as in claim 6 wherein the
selected concentration of the benzazolo[3,2-a]quinolinium chloride salt
is the IC50 of said cell.

9. A method of using the biological activity of
benzazolo[3,2-a]quinolinium chloride salts as in claim 6 wherein the time
of exposure of said cell is 48 hours.

10. A method of using the biological activity of
benzazolo[3,2-a]quinolinium chloride salts as in claim 8 wherein the time
of exposure of said cell is 48 hours.

11. A method of using the biological activity of
benzazolo[3,2-a]quinolinium chloride salts as in claim 8 wherein said
cell selected from a mammalian cell or bacterium.

12. A method of using the biological activity of
benzazolo[3,2-a]quinolinium chloride salts as in claim 8 wherein said
cell is a bacterium and said salt is used as an antibacterial.

13. A method of using the biological activity of
benzazolo[3,2-a]quinolinium chloride salts as in claim 8 wherein the
biological activity is selected from DNA fragmentation, cytotoxicity,
cell cycle disruption, generation of reactive oxygen species, and
mitochondrial permeabilization.

14. A method of using the biological activity benzazolo[3,2-a]quinolinium
chloride salts as in claim 7 wherein said cell is exposed to a
benzazolo[3,2-a]quinolinium chloride salt to differentiate between normal
and abnormal cells.

15. A method of using the biological activity benzazolo[3,2-a]quinolinium
chloride salts to differentiate cell types as in claim 14 wherein said
cell is exposed to a benzazolo[3,2-a]quinolinium chloride salt to induce
DNA fragmentation of said cell to differentiate between abnormal and
normal cells.

16. A method of using the biological activity benzazolo[3,2-a]quinolinium
chloride salts to differentiate cell types as in claim 15 wherein the
concentration of the benzazolo[3,2-a]quinolinium chloride salt is within
the range bounded by the IC25 and the IC90 of said cell.

17. A method of using the biological activity benzazolo[3,2-a]quinolinium
chloride salts to differentiate cell types as in claim 15 wherein the
concentration of the benzazolo[3,2-a]quinolinium chloride salt is the
IC50 of said cell.

18. A method of using the biological activity benzazolo[3,2-a]quinolinium
chloride salts to differentiate cell types as in claim 15 wherein the
time of exposure of said cell is 48 hours.

19. A method of using the biological activity benzazolo[3,2-a]quinolinium
chloride salts to differentiate cell types as in claim 17 wherein the
time of exposure of said cell is 48 hours.

20. A method of using the biological activity benzazolo[3,2-a]quinolinium
chloride salts to differentiate cell types as in claim 14 wherein said
cell is exposed to a benzazolo[3,2-a]quinolinium chloride salt to induce
generation of reactive oxygen species to differentiate between normal and
abnormal cells.

21. A method of using the biological activity benzazolo[3,2-a]quinolinium
chloride salts to differentiate cell types as in claim 20 wherein the
concentration of the benzazolo[3,2-a]quinolinium chloride salt is the
IC50 of said cell.

22. A method of using the physical properties of
benzazolo[3,2-a]quinolinium chloride salts to mark cells comprising: a
benzazolo[3,2-a]quinolinium chloride salt and at least one cell wherein
said cell is exposed to a selected concentration of
benzazolo[3,2-a]quinolinium chloride salt to mark said cell.

23. A method of using benzazolo[3,2-a]quinolinium chloride salts to mark
cells using the physical properties of said salts as in claim 22 wherein
said cell intakes said salt to mark said cell.

24. A method of using benzazolo[3,2-a]quinolinium chloride salts to mark
cells using the physical properties of said salts as in claim 23 wherein
said mark is a stained cell.

25. A method of using benzazolo[3,2-a]quinolinium chloride salts to mark
cells using the physical properties of said salts as in claim 24 wherein
said stained cell exhibits distinctive fluorescence.

26. A method of using benzazolo[3,2-a]quinolinium chloride salts to stain
cells using the physical properties of said salts as in claim 25 wherein
the concentration of the benzazolo[3,2-a]quinolinium chloride salt is
within the range bounded by the IC25 and the IC90 of said cell.

27. A method of using benzazolo[3,2-a]quinolinium chloride salts to stain
cells using the physical properties of said salts as in claim 25 wherein
the concentration of the benzazolo[3,2-a]quinolinium chloride salt is the
IC90 of said cell.

28. A method of using benzazolo[3,2-a]quinolinium chloride salts to stain
cells using the physical properties of said salts as in claim 25 wherein
the time of exposure of said cell is at least 4 hours.

29. A method of using benzazolo[3,2-a]quinolinium chloride salts to stain
cells using the physical properties of said salts as in claim 25 wherein
the time of exposure of said cell is between 4 and 18 hours.

30. A method of using benzazolo[3,2-a]quinolinium chloride salts to stain
cells using the physical properties of said salts as in claim 25 wherein
said cell is selected from a mammalian cell or bacterium.

31. A method of using benzazolo[3,2-a]quinolinium chloride salts to stain
cells using the physical properties of said salts as in claim 25 wherein
said method is used to measure bacterial growth.

32. A method of using the physical properties of
benzazolo[3,2-a]quinolinium chloride salts to mark organelles comprising:
a benzazolo[3,2-a]quinolinium chloride salt and at least one cell wherein
said cell is exposed to a benzazolo[3,2-a]quinolinium chloride salt to
mark at least one organelle of said cell.

33. A method of using the physical properties of
benzazolo[3,2-a]quinolinium chloride salts to mark organelles as in claim
32 wherein said mark is a stain.

34. A method of using the physical properties of
benzazolo[3,2-a]quinolinium chloride salts to mark organelles as in claim
33 wherein said stained organelle is exposed to a
benzazolo[3,2-a]quinolinium chloride salt to exhibit fluorescence of said
organelle.

Description:

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. application Ser.
No. 12/416,174, filed Apr. 1, 2009 and further claims the benefit of
priority of U.S. provisional application 61/549,209, filed on Oct. 19,
2011.

[0006] The scientific community continuously needs to apply cellular
markers when studying how cells or subjects respond or behave to a given
internal or external stimulus. Some of these stimuli include, but are not
limited to, drug treatments, therapies and natural disease processes.

[0007] U.S. Pat. No. 4,590,275 to Cox et al. (Cox '275), discloses the
preparation of benzazolo[3,2-a]quinolinium chloride salts (BQs) which
show cytotoxic, antitumor and antiviral activity. However Cox '275 fails
to disclose the synthesis of amino-substituted (ABQs) and
chloro-substituted (CBQs) benzazolo[3,2-a]quinolinium compounds, wherein
said ABQs and CBQs possess fluorescent properties applicable in the
identification of cellular organelles and biological activities
applicable for therapeutic applications. The fluorescent property assists
the user to identify cellular organelles making the compounds excellent
markers for research, diagnostic or treatment. Some of the biological
activities include, but are not limited to, cellular toxicity,
mitochondria damage and apoptosis induction on tumor cell lines in
culture.

[0008] Also Cox '275 discloses different methods for the synthesis of
several compounds; however the disclosed methods do not limit the
excitation by irradiation of the compounds in order to avoid unwanted
reactions resulting in unwanted characteristics and properties. Further
in Cox '275 the BQs were isolated as the perchlorate salt through its
precipitation by addition of an aqueous perchloric acid or a saturated
sodium perchlorate solution avoiding isolating the pure BQs.

SUMMARY OF THE INVENTION

[0009] The present invention overcomes the limitations of the synthesized
benzazolo[3,2-a]quinolinium chloride salts as presented by Cox '275 et
al. and discloses a synthesis procedure that modifies the biological
activity and greater selectivity toward tumor cells providing the
autofluoresce in cell cultures, inducing cytotoxity through apoptosis,
binding to cellular organelles and caspases activation on human tumor
(abnormal) or normal cells in culture. The term "modify", or its
derivations, as used throughout this specification and the claims when in
reference to biological activity, is defined as follows: to make the
form, nature, content, or future course of the biological activity
different from what the biological activity is or from what the
biological activity would have been if not exposed to the referenced
compound.

[0010] First, the present invention discloses several compounds, such as
amino-substituted benzazolo[3,2-a]quinolinium showing (1) DNA
fragmentation; (2) cell cycle disruption; (3) cellular toxicity; and (4)
generation of reactive oxygen species (ROS). Also the compound has shown
auto-fluorescent properties in contact with cells. These fluorescent
properties allow a clear indication of the interaction with cellular
organelles serving as a fluorescence marker in research or as a
therapeutic marker or a diagnostic marker in clinical studies. It could
be applied to monitor the presence and concentrations of microorganisms
in the environment since they also possess cellular organelles to which
these fluorescent compounds can bind. Further the ABQs display increased
selectivity against cancer cells as compared to NBQs.

[0011] Second, the present invention discloses the synthesis of
chloro-substituted and amino-substituted benzothiazolo[3,2-a]quinolinium
salts causing cell death via an apoptosis mechanism, interaction with
macro organelles such as mitochondria and DNA, activation of caspases 3
and 7, and the formation of 8-2-dG adducts upon bioreduction in the
presence of XO/HX.

[0012] Third the present invention discloses an improved procedure for the
synthesis of benzothiazolo[3,2-a]quinolinium salts (BQs).

[0013] Therefore one of the objectives of the present invention is to
provide a compound showing DNA fragmentation, cell cycle disruption,
cellular toxicity, generation of reactive oxygen species, and auto
fluorescence.

[0014] Another object of the invention is to provide a synthesized
nitro-substituted, amino-substituted, and chloro-substituted
benzothiazolo[3,2-a]quinolinium salts (BQs) causing cell death via an
apoptosis mechanism, interaction with macro organelles such as
mitochondria and DNA, activation of caspases 3 and 7, and the formation
of 8-2-dG adducts upon bioreduction in the presence of XO/HX.

[0015] Another objective of the invention is to provide a synthesized
nitro-substituted amino-substituted, and chloro-substituted
benzothiazolo[3,2-a]quinolinium salts that allows the identification of
tumor cells.

[0016] Another objective of the invention is to provide a new improved
method for the synthesis of BQs which is more compatible with the
biological systems.

[0017] Another objective of the invention is to provide a synthesis method
of BQs that avoids unwanted reactions.

[0018] Another objective of the invention is to provide a synthesis method
of BQs that improves performance of the compounds.

[0019] Another objective of the present invention is to provide a compound
suitable as an anti-cancer therapeutic agent, bacterial marker, and
antibacterial.

[0020] The invention itself, both as to its configuration and its mode of
operation will be best understood, and additional objects and advantages
thereof will become apparent, by the following detailed description of a
preferred embodiment taken in conjunction with the accompanying drawing.

[0021] The Applicant hereby asserts, that the disclosure of the present
application may include more than one invention, and, in the event that
there is more than one invention, that these inventions may be patentable
and non-obvious one with respect to the other.

[0022] Further, the purpose of the accompanying abstract is to enable the
U.S. Patent and Trademark Office and the public generally, and especially
the scientists, engineers, and practitioners in the art who are not
familiar with patent or legal terms or phraseology, to determine quickly
from a cursory inspection the nature and essence of the technical
disclosure of the application. The abstract is neither intended to define
the invention of the application, which is measured by the claims, nor is
it intended to be limiting as to the scope of the invention in any way.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings, which are incorporated herein,
constitute part of the specifications and illustrate the preferred
embodiment of the invention.

[0024] FIG. 1 Shows the structure of a preferred embodiment of the present
invention.

[0037] FIG. 1 shows a general structure of the synthesized NBQs and ABQs.
The present invention is particularly directed to the synthesis and
biological activity of ABQs as shown in FIG. 2. The synthetic sequence is
disclosed in patent application Ser. No. 12/416,174, included here by
reference.

[0038] The particular methodology or method comprises several measurements
and apparatus used in order to perform the synthesis of the compounds.
The BQs, NBQs and ABQs, were synthesized using several methods as
disclosed in patent application Ser. No. 12/416,174 herein included by
reference.

[0039] The synthesis of benzazolo[3,2-a]quinolinium chlorides are
performed using at least four different methods, as disclosed in patent
application Ser. No. 12/416,174 herein included by reference.

[0040] Further a green procedure for the synthesis of
benzazolo[3,2-a]quinolinium salts (Method 5) was developed. The
photochemical cyclization and work-up procedure for the synthesis of
benzazolo[3,2-a]quinolinium salts was described in the first application
Ser. No. 12/416,174 as Methods 1, 2, 3, or 4. New Method 5 is partially
similar as described before, except that the corresponding
(E)-2-(2-chlorostyryl)benzazole was dissolved in 150-250 mL of a 2:2:1
heptane (or octane):dioxane:bromobenzene mixture. The elimination of
benzene as a solvent constitutes an environmentally friendlier or green
chemical procedure. The following is a representative example of the use
of Method 5.

Representative Example to Validate the Modified Method

[0041] 7-benzyl-3-nitrobenzimidazolo[3,2-a]quinolinium chloride (NBQ-48).
Photochemically induced cyclization of
(E)-7-benzyl-2-(2-chloro-3-nitrostyryl)benzimidazole as described in
Method 5 produced the title compound, in 70% yield, identical in all
respects to that of a sample obtained using Method 1 of patent
application Ser. No. 12/416,174.

[0068] The inclusion of the chloro-substituents, amino-substituents, and
nitro-substituents in ring A to the synthesis of (E)-2-styrlbenzazole and
benzazolo[3,2-a]quinolinium chlorides (BQs) results in ABQs and NBQs
providing several properties, more particularly exhibiting an increment
in the biological activity. For example FIG. 3 provides the
characterization of the representative the ABQ48.

[0069] FIGS. 4 and 5 show the fluorescent chloro-substituted
benzazolo[3,2-a]quinolinium salts. This new series of halogen substituted
benzazolo[3,2-a]quinolinium salts; without nitro or amino substituents
display relatively high fluorescence intensity in aqueous solution (see
FIG. 4) thus making them potentially useful cellular markers similar to
ABQs. For example 4-chloro-10-methylbenzothiazolo[3,2-a]quinolinium
chloride (GHQ-108) displays good florescence intensity and cytotoxicity
activity against A-431 cell line (IC50 3.7 μM).

[0078] Reduction in the cell viability of the three cell lines after 24
and 48 hours treatments was observed. Higher susceptibility was observed
by Tk6 lymphoblasts with an average IC50 of 8.6 μM in contrast to
Toledo cells with an IC50 of 50 μM which were the least
susceptible cultures to the drug. The IC50 of the A431 (epitheloid
carcinoma) cells was 23 μM (FIG. 6) which is comparable to other
previously studied BQs compounds of this family (Vivas--Mejia, 1998;
Arroyo and Zayas, 2007; unpublished data). FIG. 1 presents the
comparative dose response curve and IC50 for ABQ 48 in the three
cell lines. Mitochondrial membrane permeability, DNA fragmentation and
cell cycle alteration was also observed in the three cell lines.

[0079] Bioactivity 2: Time dependent uptake and retention of ABQ48 on
cells up to 24 hours

[0080] This assay aims to determine the uptake and retention of the
fluorescence ABQ48 on three cell lines as a function of time. The cell
lines used were A431 epitheloid carcinoma, Toledo lymphoma and Tk6 normal
lymphoblast.

[0084] Methods--A431 epitheloid carcinoma, Toledo lymphoma and Tk6 normal
lymphoblast cultures were exposed to ABQ 48 and incubated at 37°
C. and 5% CO2 for 0, 1, 4 and 18, hours at their IC90 dose (90
μM, 100 μM, and 135 μM respectively). A non-exposed negative
control (analytical grade water) for each determined time point was also
included. Cells were then washed twice with PBS (Sigma, St. Louis Mo.)
and their fluorescence in standard units (FSU) measured using a Modulus
fluorometer (Promega, Sunnyvale, Calif.). Triplicate measures were
obtained from each sample. Values were then averaged and background
corrected using the negative control values. A one way ANOVA with fixed
effects was performed; in case significant differences were found in
ANOVA, a Post Hoc Test Tukey Honestly Significant Difference (HSD) was
also performed using SPSS software (IBM, Armonk, N.Y.).

[0085] Results and Discussion

[0086] The time dependent drug uptake assay clearly demonstrated an
increment in the fluorescent emission of cells from three different cell
lines (A931, Toledo, and TK6) when treated with ABQ48 for 0 hour, 1 hour,
4 hours, and 18 hours indicating the uptake and retention of A98 in the
cells. Results were measured in Fluorescence Standard Units (FSU) versus
time of exposure (FIG. 8-10).

[0087] Bioactivity 3. DNA fragmentation--Fragmentation of DNA as a marker
for apoptosis is a commonly used assay. Analysis was performed applying
the Nucleo counter NC3000 DNA fragmentation assay. Determination of the
degree of DNA fragmentation is based on the retention of DAPI, a nucleic
acid staining reagent. As described previously TK-6, and Toledo cells
were kept in culture at the specified conditions. Cells where then
treated with the tested compounds as previously described for 48 hours,
after which nutritional media removed, cells harvested, fixed with 70%
ethanol, incubated and stained with 1 μg/ml DAPI according to
manufacturer's specifications for image analysis measuring DAPI
intensity.

[0088] Results and Discussion

[0089] As presented in FIG. 11 cells treated with ABQ48 and NBQ48
presented DNA fragmentation at different levels depending on the cell
type, but higher than the negative control population.

[0090] Bioactivity 4. Cell Cycle disruption--Cell cycle effects are of
great importance to the characterization of any novel therapeutic agent.
This most fundamental cellular process is integral in determining the
effects of any drug upon the target cells. After treatment with the
tested BQs at previously described conditions, cells were harvested, and
stained with 10 μg/ml DAPI (a DNA marker) according to manufacturer's
specifications and analyzed with the instrument using image analysis to
measure the DAPI intensity as an indicator of the DNA density.

[0091] Results and Discussion

[0092] As presented in FIG. 12 the tested ABQ48 induces changes in the
cell cycle of the treated cells in comparison with the negative control
(none treated cells). Most cells treated with ABQ 48 are arrested at the
S/G2 stage in contrast with the control population where cells are
distributed among the cells cycle stages.

[0093] The biological activities such as generation of DNA fragmentation
and cell cycle disruption observed in cells treated with ABQ48 as
discussed in Bioactivity 3 and Bioactivity 4 have been observed at
different degrees with ABQ 95, ABQ 38, NBQ 95 and NBQ38 on the A431 and
TK6 cell lines. The applied method is as reported above:

[0094] DNA fragmentation--Analysis was performed applying the Nucleo
counter NC3000 DNA fragmentation assay. Determination of the degree of
DNA fragmentation is based on the retention of DAPI, a nucleic acid
staining reagent. Prior to treatment with the tested compounds A431 and
TK-6 cells were kept in culture at the specified conditions. Cells where
then treated with the tested compounds at the respective IC50
concentrations (between 25 and 300 μM). DNA fragmentation protocol was
as recommended by Nucleo counter NC3000 manufacturer.

[0095] Results and Discussion

[0096] The tested compounds presented little DNA fragmentation in the
tested cell lines. Over all in tumor cells, such as A431, BQs 95 induced
fragmentation at levels close or similar to the negative control in
contrast to BQs 38. In TK6 low DNA fragmentation was observed as well.

[0097] Cell Cycle disruption--Cell cycle effects are of great importance
to the characterization of any novel therapeutic agent. After treatment
with the tested BQS 95 and BQS 38 at previously described conditions,
cells were harvested, and stained with 10 μg/ml DAPI (a DNA marker)
according to manufacturer's specifications and analyzed with the
instrument using image analysis to measure the DAPI intensity as an
indicator of the DNA density.

[0098] Results and Discussion

[0099] Changes in the cell cycle of A431 and TK6 where observed at a
higher level with the NBQ 38 than with NBQ 95 with little to none cell
cycle effects of the treated cells in comparison with the negative
control (none treated cells). Most cells treated with ABQ 48 are arrested
at the S/G2 stage in contrast with the control population where cells are
distributed among the cells cycle stages.

[0100] Reactive Oxygen Species (ROS)--An increase in the generation of
Reactive Oxygen Species (ROS) can indicate an apoptotic event involving
damage to mitochondria. For determination ROS generation the fluorescent
dye 2,7-dichlorofluorescein diacetate (DCFH-DA) was applied a standard
protocol based on Park and Park 2007. In this experiment A431 and TK6
cells where treated with ABQ38, ABQ95, NBQ 38 and NBQ 95 at their
respective IC50.

[0101] Results and Discussion

[0102] A clear and strong induction of the generation of ROS was observed
in A431 treated with ABQ38, ABQ95, NBQ 38 and NBQ 95. In TK6 however, the
generation of ROS was close to the level observed in the negative
control.

[0103] Marker of bacterial growth and antibacterial--The biological
activities of the BQs, for example ABQ 48 is further used as a marker of
bacterial growth and antibacterial. The fluorescent properties of ABQ 48
an unnatural amino substituted alkaloid with fluorescent properties where
applied upon bacterial cells. Two bacterial cell lines Escherichia coli
(Gram negative) and Staphylococcus aureus (Gram positive) were maintained
at logarithmic growth in nutrient broth media at 37° C. (NB) and
were exposed to 150 μM dose of ABQ 48 for periods of 2, 4 and 6 hours.
Cells were washed twice with PBS (Sigma, St. Louis, Mo.) at 2500 rpm and
resuspended in 500 μL. Fluorescence in standard units (FSU) was
measured using a Modulus fluorometer (Promega, Sunnyvale, Calif.). Three
measurements were obtained from each sample. Values were then averaged
and background corrected using the negative control values. Results
indicate that Gram negative cells fluoresce faster than gram negative.
After a 2 hour exposure Gram negative stained as observed by an increase
in the fluorescence in standard units (FSU) in contrast to Gram positive
cells which fluoresce after a 4 hour exposure.

[0104] Use of ABQs as antibacterial is based on a standard Kirby-Bauer
antimicrobial susceptibility test where the growth inhibition of a known
bacterial colony in the presence of the ABQS is measured.

[0105] BQs drug-likeness analysis--FIGS. 13 and 14 include the
drug-likeness and Mol LogP of ABQ-2, 38, 48, 91 and 95 and other
compounds. ABQ-95 displays the highest drug-likeness (0.43) among those
already reported in Ser. No. 12/416,174. Log P is the Log of the
partition of the compound between an organic phase (e.g. octanol) and an
aqueous phase (e.g. buffer) at a pH. BQs are lipophilic cations, and a
correlation has been suggested between Log P and the preference of this
type of compounds to interact with the mitochondria. Therefore, it is of
interest to correlate mitochondrial permeabilization, one of the
bioactivities we have determined for ABQs, and Mol Log P. Results shown
in FIG. 13 and FIG. 14 serves as a guide to design and synthesize
compounds with increased drug-likeness and their Mol Log P.

[0106] The invention is not limited to the precise configuration described
above. While the invention has been described as having a preferred
design, it is understood that many changes, modifications, variations and
other uses and applications of the subject invention will, however,
become apparent to those skilled in the art without materially departing
from the novel teachings and advantages of this invention after
considering this specification together with the accompanying drawings.
Accordingly, all such changes, modifications, variations and other uses
and applications which do not depart from the spirit and scope of the
invention are deemed to be covered by this invention as defined in the
following claims and their legal equivalents. In the claims,
means-plus-function clauses, if any, are intended to cover the structures
described herein as performing the recited function and not only
structural equivalents but also equivalent structures.

[0107] All of the patents, patent applications, and publications recited
herein, and in the Declaration attached hereto, if any, are hereby
incorporated by reference as if set forth in their entirety herein. All,
or substantially all, the components disclosed in such patents may be
used in the embodiments of the present invention, as well as equivalents
thereof. The details in the patents, patent applications, and
publications incorporated by reference herein may be considered to be
incorporable at applicant's option, into the claims during prosecution as
further limitations in the claims to patently distinguish any amended
claims from any applied prior art.